Prior to a discussion of the background art, it is noted that the bracketed [ ] numbers in the discussion refer to the enumerated references in the Bibliographical References listed below at the end of the specification.
With respect to the background art, in the U.S.A., prostate cancer is the most frequently diagnosed form of cancer and the second leading cause of cancer death in males [2]. Current modalities of therapy for localized tumors include surgery and radiotherapy, and are generally successful. However, treatment for metastatic disease is not as beneficial, because current hormonal therapies work only transiently [3]. Therefore, new treatments for prostate cancer are needed.
Immunotherapy, based on CD8+ cytotoxic T lymphocytes (CTL) is one potential new avenue of therapy that holds much promise, especially for prevention and adjuvant treatment of metastatic disease [4,5]. CTLs recognize antigen in the form of a short peptide (8-10 amino acids) in a complex with class I major histocompatibility complex (MHC) on the surface of target cells. The ability of CTL to directly lyse these cells makes them attractive for tumor immunotherapy.
Prostate-specific antigen (PSA) has been proposed as a tumor antigen for the specific destruction of prostate carcinoma cells by CTLs. Tight tissue specificity of expression to the prostate, continued expression by prostate carcinoma cells, and the wealth of biochemical, genetic, and cell biological data available all make PSA an excellent candidate for characterization as potential target for prostate cancer immunotherapy.
Several PSA-based vaccines were evaluated in recently conducted clinical trials for stimulating an immune response against PSA in patients with advanced prostate cancer. These vaccines represented a recombinant vaccinia virus expressing PSA (rV-PSA) [6-9], a recombinant PSA protein formulated in liposomes [10], and autologous dendritic cells (DCs) pulsed with recombinant PSA protein [11] or transfected with PSA-encoding RNA [12].
CD8+ T Cells
The main biological function of CD8+ T cells is to eliminate pathogen-infected cells in the body. The mechanism responsible for T-cell recognition of infected cells is now well established at the molecular level and relies on interaction between a T-cell receptor complex (TCR) and an antigen-derived peptide bound to a major histocompatibility complex class I molecule (MHC I). All protein antigens produced by the cell are eventually degraded and the resulting peptides are presented by MHC I molecules on the cell surface.
Development of CD8+ T Cells
Development of T cells occurs in the thymus, where TCR α and β gene segments are rearranged such that each T cell clone eventually expresses a unique TCR [13]. Developing thymocytes that produce a surface TCR express CD4 and CD8 co-receptors and undergo a complex process of maturation, depending on the specificity and affinity of their TCRs for self-peptide MHC ligands. Thymocytes that express TCRs with no affinity for self-peptide-MHC molecules die by a programmed cell death mechanism. Potentially harmful thymocytes that express TCRs with strong affinity for the self-peptide-MHC ligands expressed on cells in the thymus are eliminated via physical deletion [14], functional inactivation [15], or receptor editing [16]. Only thymocytes that express TCRs with a low but significant affinity for self-peptide-MHC ligands on thymic stromal cells survive thymic selection [17].
Recirculation and Survival of Naive CD8+ T Cells
T cells that have not yet encountered a foreign peptide-MHC ligand for which their TCR has a high affinity are referred to as “naive” T cells. These cells account for the majority of T cells in the secondary lymphoid organs in healthy young adults. Naive T cells recirculate continuously through the secondary lymphoid organs, which include spleen, lymph nodes, and mucosal lymphoid organs (such as Peyer's patches of the intestines) [18, 19]. It is estimated that an individual naive T-cell will on average circulate through the secondary lymphoid organs for several months [20, 21]. Survival of naive CD8+ T cells during this normal lifespan is maintained by low-affinity TCR recognition of self-peptide-MHC complexes [22] and signaling through the IL-7 receptor [23, 24]. Although signals through the TCR and IL-7 receptor are required for the survival of naive T cells, these signals do not cause the T cells to proliferate in hosts containing normal numbers of T cells. In contrast, naive T cells proliferate when transferred into T-cell-deficient hosts. This “homeostatic” proliferation also depends on IL-7 [23, 24] and low-affinity TCR recognition of self-peptide-MHC complexes [25], but not IL-2 or the CD28 co-stimulatory receptor [26]. In young individuals, new naive T cells are constantly produced by the thymus and exported to the secondary lymphoid organs to replace senescent naive T cells. In contrast, in older individuals whose thymic output is reduced or absent, senescent cells may be replaced by proliferation of remaining naive T cells.
Activation of CD8+ T Cells
Naive CD8+ T cells migrating through the T-cell areas of secondary lymphoid organs encounter a dense network of large, irregular shaped dendritic cells (DCs) that constitutively express the highest levels of MHC molecules of any cell in the body (271. In the absence of infection or tissue damage, all DC populations in the secondary lymphoid organs exist in a resting state characterized by low expression of co-stimulatory molecules such as CD80 and CD86 [28]. In this state, DCs most likely play an important role in the presentation of low-affinity self-peptide-MHC ligands that maintains survival of naive T cells.
In the case of infection, various viral or bacterial products are recognized by pattern recognition receptors [29], for example, Toll-like receptors (TLRs) on cells of the innate immune system, including DCs. TLR signaling causes activation of DCs, which results in expression of higher levels of co-stimulatory molecules (CD80 and CD86) and production of inflammatory cytokines [30]. Activated DCs then function by presenting pathogen-derived peptide-MHC class I complexes to naive CD8+ T cells. In addition to a signal through TLR, naive CD8+ T cells also require additional signals through the co-stimulatory CD28 receptor and the IL-12 receptor to proliferate maximally and differentiate into cytotoxic effector cells [31-33]. All these signals can be provided to naive CD8+ T cells by activated DCs [34].
Naive CD8+ T cells show signs of DNA replication and cell division as early as 48 hours after exposure to antigen in vivo [35-37]. These events are followed by an exponential increase in the number of antigen-specific T cells over the next several days. Depending on the stimulus, the number of antigen-specific CD8+ T cells reaches its highest level in the secondary lymphoid organs, 7 to 15 days after activation with an antigen (FIG. 2,3) [35, 38-43].
In vitro experiments indicate that cell division by naive, antigen-stimulated T cells is driven by autocrine production of IL-2 [44]. Surprisingly, however, antigen-driven proliferation of naive T cells is minimally dependent on IL-2 in vivo [45-49]. Therefore, in addition to IL-2, other signals or growth factors must be also capable of driving T-cell proliferation in vivo.
In vivo T-cell proliferation is tightly regulated by co-stimulatory signals from DCs. The proliferation of antigen-stimulated CD8+ T cells is reduced dramatically in mice in which CD28 cannot interact with its ligands CD80 and CD86 [37, 45, 50]. CD40 ligand deficiency has a similar effect on T-cell expansion, which may be related to the fact that CD40 signaling induces CD80 and CD86 on antigen-presenting cells [51]. Co-stimulatory signals regulate T-cell proliferation by enhancing growth factor production. Antigen-driven IL-2 production is greatly impaired when CD28 signaling is eliminated [45].
Effector CD8+ T Cells
Antigen-specific CD8+ T cells at the peak of immune response express effector functions, and thus are sometimes referred to as “effector cells” [52]. Effector cells express a characteristic set of adhesion receptors. Unlike naive cells they express perforin and granzymes, which contribute to their defining feature, that is, the ability to directly kill target cells that display the appropriate peptide-MHC class I complexes. [53]. The effector T cells migrate out of the T-cell areas and into many nonlymphoid tissues, particularly inflamed sites of antigen deposition. The migration of effector CD8+ T cells with cytolytic potential into nonlymphoid organs is an effective way of eliminating cells that display peptide-MHC class I complexes from all parts of the body.
The number of effector T cells in the secondary lymphoid organs falls dramatically after the peak of proliferation [35, 38-43]. The molecular basis for death of effector T cells varies depending on the nature of the antigenic stimulus. In the case of a T cell response after a single administration of antigen, the death is Fas-independent and Bcl-2 sensitive [54] and occurs most likely due to deprivation of growth factors [55]. If antigen is presented chronically, TCR-mediated activation-induced cell death (AICD) may occur [56]. This type of apoptosis is dependent of Fas and is poorly inhibited by Bcl-2 [55].
IL-2 is playing a role in the AICD by preventing the activation of FLICE inhibitor protein, which normally inhibits Fas signaling [57]. The death of effector CD8+ T cells is regulated by inflammation. In the absence of inflammation, the loss of antigen-specific T cells from the secondary lymphoid and nonlymphoid organs after the peak of proliferation is nearly complete [58]. In contrast, many more cells survive the loss phase after injection of antigen together with adjuvants such as LPS or IL-1 [35, 58, 59].
Memory CD8+ T Cells
The vast majority of effector cells die after the peak of proliferation, nevertheless, a stable population of antigen-experienced T cells survive for long periods of time if the antigen was initially presented in an inflammatory context [52]. In many ways, memory cells can be thought of as effector cells that have returned to a basal activation state. Indeed, several lines of evidence suggest that effector cells are precursors of memory cells [60, 61].
Unlike naive CD8+ T cells, memory CD8+ T cells do not depend on MHC-class 1 molecules for survival [62]. Whereas most memory CD8+ T cells are not cycling, a small fraction of the memory population is proliferating in an MHC class I-independent fashion at all times [47, 62]. This proliferation is balanced by death since the total number of antigen-specific memory CD8+ T cells remains unchanged over time. Several observations suggest that IL-15 plays a role in this process. The antigen-independent proliferation of memory CD8+ T cells is accelerated by injection of IL-15 [63] and blocked by injection of antibodies against IL-15 [47]. In addition, memory CD8+ T cells are diminished in IL-15-deficient mice [64]. Since IL-15 is produced by non-T cells during the innate immune response, it is possible that memory CD8+ T cells are maintained as a consequence of IL-15 produced in response to other infections [63, 65].
Prostate-Specific Antigen (PSA)
Prostate-specific antigen (PSA) is a kallikrein-like, serine protease that is produced exclusively by the columnar epithelial cells lining the acini and ducts of the prostate gland [66-68]. PSA is secreted into the lumina of the prostatic ducts and is present in the seminal plasma at rather high concentrations ranging from approximately 0.5 to 5 mg/ml [69]. Physiologically, PSA functions in seminal plasma to cleave the major gel-forming proteins semenogelin I and II, and fibronectin, resulting in increased sperm motility [67, 70, 71]. PSA is translated as an inactive 261 amino acid preproPSA precursor. PreproPSA has 24 additional residues that constitute the pre-region (the signal peptide) and the propeptide. Release of the propeptide results in the 237-amino acid, mature extracellular form, which is enzymatically active. Human glandular kallikrein 2 (hKLK2), which like PSA is preferentially expressed in the prostate tissue, is responsible for the activation of proPSA [72]. PSA has been shown to contain an N-linked oligosaccharide attached to asparagine-69 [73].
PSA is also released into the blood at low concentrations. In healthy males without clinical evidence of prostate cancer, the concentration of PSA detected in the serum is usually less than 4 ng/ml [74-76]. Enzymatically active PSA is inactivated in the blood by forming covalently linked complexes with α1-antichymotrypsin (ACT) [77, 78]. Enzymatically inactive (internally clipped) PSA is incapable of forming complexes with protease inhibitors and circulates as a free, uncomplexed form in the blood [79].
PSA is organ-specific and, as a result, it is produced by the epithelial cells of benign prostatic hyperplastic (BPH) tissue, primary prostate cancer tissue, and metastatic prostate cancer tissue [66, 80]. Normal prostate epithelial cells and BPH tissue actually produce more PSA protein than malignant prostate tissue [81, 82]. Therefore, PSA is not a traditional tumor marker that is produced in higher quantities by tumor cells, but rather abnormalities in the prostate gland architecture resulting from trauma or disease can lead to increased “leakage” of the enzyme into the stroma and then into the bloodstream via capillaries and lymphatics.
The most common use of PSA in the clinic is for monitoring prostatic cancer therapy. If a patient undergoes a radical prostatectomy, serum PSA levels should decrease to undetectable concentrations because all of the source tissue has been removed [83, 84]. Increasing PSA concentrations after surgery, indicate a recurrence of the disease [83, 85, 86]. PSA also reflects the success of radiotherapy and anti-androgen (hormonal) therapy in prostate cancer patients [87-89].
Plasmid DNA Vaccines Against Cancer
In recent years a number of tumor vaccination strategies have been developed. Most of them rely on identification of tumor antigens that can be recognized by the immune system. DNA vaccination represents one such approach for the induction of both humoral and cellular immune responses against tumor antigens. Studies in animal models demonstrated the feasibility of using DNA vaccination for eliciting protective anti-tumor immune responses. However, most tumor antigens expressed by cancer cells in humans are weakly immunogenic, which requires development of strategies to potentiate DNA vaccine efficacy in the clinical setting. Recent advances in understanding the immunology of DNA vaccines and strategies used to increase DNA vaccine potency with respect to CTL activity are discussed below.
Immunology of DNA Vaccines
A DNA vaccine usually represents a simple plasmid DNA expression vector. It contains cDNA encoding a desired antigen inserted between a eukaryotic promotor and a polyadenylation sequence, bacterial antibiotic resistance gene and a bacterial origin of replication. The eukaryotic promotor and polyadenylation sequence are required for proper antigen expression in mammalian cells, and the antibiotic resistance gene and origin of replication allow production of the vector in bacteria.
After administration of the naked plasmid DNA by intramuscular (i.m.) or intradermal (i.d.) inoculation, host cells take up the DNA and produce the encoded antigen, which then serves as a target for the immune response [90-93]. The expression of the antigen in vivo is commonly achieved by using strong viral promoters, which are ubiquitously active and will drive antigen production in a wide range of cell types. The human cytomegalovirus immediate early enhancer-promotor (known as the CMV promotor) is often the promotor of choice [94]. DNA vaccination results in generation of adaptive immune responses comprising of regulatory components such as: induction of antigen-specific CD4+ helper T cells; and effector components such as: production of antibodies recognizing native antigen, and effector CD8+ cytotoxic T lymphocytes (CTLs). The latter are directed against antigen-derived peptides presented by class I major histocompatibility molecules (MHC class I) on the cell surface.
The potential of DNA encoding a protein antigen to generate CTL responses has attracted a lot of attention, since immunization with purified recombinant proteins does not efficiently induce CTLs (reviewed in [95, 96]). Studies in mice of the underlying mechanism revealed that induction of helper CD4+ T cells and direct activation of antigen-presenting cells (APCs) by DNA molecules contributes to the successful CTL priming by DNA vaccines. The latter requirement was suggested to be somewhat redundant (see below).
Here we summarize the key findings, which are starting to elucidate the observed immunogenicity of DNA vaccines.
The CD8+ T-cell response after DNA vaccination was shown to be initiated by bone marrow-derived APCs, such as dendritic cells (DCs) [97-99]. The relevant APCs can be directly transfected with plasmid DNA, which then leads to antigen production within the cell, or they may pick up antigen expressed and released by other cells (the latter mechanism is referred to as cross-presentation/cross-priming) [100-102]. In both cases, the antigen is processed by proteolytic digestion inside the APCs and the resulting peptides are presented by MHC class 1 molecules on the cell surface for priming of naive CD8+ T cells. Which of these two mechanisms is the predominant one in vivo is still a matter of a debate and may vary among different DNA administration methods [102-104].
Nevertheless, certain modifications of the antigens (including linkage with ubiquitin [105] or heat shock proteins [106, 107]) may improve their targeting to the conventional or cross-priming MHC class I presentation pathway (reviewed in [108]).
The backbone of plasmid DNA was shown to contain immunostimulatory nucleotide sequences, which are composed of unmethylated CpG dinucleotides, with particular flanking nucleotides (referred to as CpG motifs) [109-111]. Due to differences in frequency of utilization and methylation pattern of CpG dinucleotides in eukaryotes versus prokaryotes, such sequences are approximately 20 times more common in bacterial then in mammalian DNA [112, 113]. The CpG motifs were shown to act through Toll-like receptor 9 (TLR9) [114], which is expressed in mice on macrophages, DCs and B cells, but in humans only on plasmacytoid DCs and B cells [115-117]. The direct interaction of TLR9 and CpG-containing plasmid DNA was shown to result in upregulation of co-stimulatory molecules on APCs and induction of the proinflammatory cytokines IL-12, IL-6, IL-18, TNFα, IFNα/β and IFNγ, secreted by various cells of innate immune system [118-121].
Activation of APCs is known to be important for efficient priming of naive CD8+ T cells and thus presence of certain CpG motifs in the backbone of DNA vaccines was suggested to contribute to CTL induction [122]. Surprisingly, repeated DNA immunization of mice with deficiency in the TLR9 signaling pathway (TLR9−/− or MyD88−/− mice) results in development of normal CTL responses, similar to those in wild-type mice, despite the fact that no direct stimulation of APCs by plasmid DNA could be observed in these mice [123, 124]. This finding suggests that activation of APCs by CpG-motifs might be redundant in the context of DNA vaccines, and necessary activation of APCs in vivo can possibly occur indirectly, through induction of helper CD4+. T cells (reviewed in [125]). In fact, DNA immunization experiments in mice either depleted of CD4+ T cells or having deficiency in CD4+ T cell compartment (CD4−/− or MHC class II−/− knockouts) demonstrated that the presence of CD4+ T cells is a critical requirement for generation of effector CTL responses [126-128].
DNA Vaccines Against Cancer in Animal Models
The utility of DNA vaccines in developing protective anti-tumor responses was first demonstrated with model tumor antigens in mice. DNA immunization with plasmids encoding the SV40 large T-antigen [146], β-galactosidase [147], human carcinoembryonic antigen (CEA) [148], human papillomavirus E7 [149] or human PSA [150] were shown to protect mice from lethal challenge with syngeneic tumor cells expressing the corresponding antigen. Depletion studies provided evidence for the role of CD8+ cytotoxic T lymphocytes in the tumor rejection [147, 149]. Altogether these studies demonstrate the feasibility of using DNA vaccines for inducing antigen-specific immune responses targeting tumor cells. However, all of the antigens used in these studies were in fact foreign proteins which typically are much more immunogenic than the “regular” tumor antigens which represent self-antigens.
Several murine models were established to allow testing of DNA vaccine potency against tumor antigens that more closely resemble those that would be encountered clinically. These approaches rely on the use of transgenic mice expressing model tumor antigens in a tissue specific manner [151, 152], or testing DNA vaccines that target the murine counterparts of human tumor antigens [153, 154].
DNA immunization against the P815A antigen, a murine equivalent of human tumor-specific antigens belonging to the MAGE family [155], was shown to induce CTLs and protect mice from lethal tumor challenge [153]. This finding suggested that the T cells could be readily induced against natural tumor-specific antigens, which are silent in most normal tissues.
In contrast, naturally occurring tumor-associated antigens were shown to have low intrinsic immunogenicity. While a DNA vaccine encoding human proto-oncogene Her2 readily induced an antibody response in wild-type mice, the same vaccine induced only a modest antibody response in Her2 transgenic mice and provided weak tumor protection [152]. Similar results were also obtained for CTL responses in Her2/neu transgenic mice. Immunization with the rat neu DNA vaccine induced protective CTL responses in wild-type mice, but was not effective in transgenic animals where no CTL response was observed [156]. The ability of the rat neu DNA vaccine to induce CTL responses in wild-type mice could probably be explained by the differences in the amino acid sequence between the neu-derived CTL epitope and the corresponding sequence of the murine Her2 counterpart (c-erbB-2) [156, 157]. Thus, CD8+ T cells capable of recognizing the neu-derived epitope are present in wild-type mice, but are most likely deleted during thymic selection or anergized in the periphery in neu-transgenic animals.
In line with these findings, DNA immunization against murine melanocyte differentiation antigens TRP-1, TRP-2 (tyrosinase-related proteins), and gp100 were also unsuccessful [154, 158, 159]. Interestingly, the same studies demonstrated that immunization of mice with the xenogeneic (human) DNA encoding TRP-1, TRP-2, or gp100 resulted in induction of immune responses and protection from syngeneic tumor challenge with B16 mouse melanoma cells. The anti-tumor immunity was mediated by antibodies upon vaccination with human TRP-1, and by CD8+ T cells in the case of human TRP-2 and gp100 (reviewed in [160]) A significant conclusion of these observations is that immunization with syngeneic (mouse) genes does not induce T-cell or antibody responses, while immunization with xenogeneic (human) genes can lead to the generation of antibodies and CTLs capable of recognizing both the human and mouse proteins. For CTL responses, the mechanism underlying such cross-reactivity was shown, in case of gp100, to represent the random creation of a heteroclitic epitope in the human sequence with better binding capacity to a MHC class I antigen [161]. Thus, a DNA vaccine encoding human gp100 induces CD8+ T cells that are directed against this “human” epitope and are also capable of recognizing the corresponding murine endogenous sequence (“murine” epitope) [159, 162]. For cross-reactive antibody responses, the presence of strong helper epitopes within the xenogeneic sequence was suggested [163]. To this end, research on DNA vaccines in animal models have shown some promising results regarding tumor protection. Challenges remain, however, for the use of DNA vaccines as a therapeutic tool, which is more reflecting the clinical setting. Further understanding of the mechanisms underlying the formation of the T cell repertoire during T cell maturation in the thymus and exact mapping of epitope specificity for “self” tumor antigen reactive CTLs, should provide further help for the rational design of DNA vaccines capable of inducing more potent immune responses particularly against tumor-associated antigens.
Enhancing Potency of DNA Vaccines
Studies in mice have demonstrated that the frequencies of antigen-specific CTLs induced by DNA vaccines are around 10-fold lower when compared to virally induced responses, and the primary effector CTL response after a single DNA immunization is slightly delayed, peaking at 12-15 days after immunization [164, 165]. These qualitative differences in primary CTL responses could be in part attributed to the minute amounts of antigen produced after plasmid DNA administration [90] and inefficient targeting of APCs in vivo, which altogether is not sufficient to ensure robust priming and expansion of naive T cells.
Several approaches for DNA delivery have been developed which provide elevated amounts of antigen produced and/or improved targeting of APCs in vivo, when compared to the commonly used i.m. or i.d. injection of DNA in saline. These techniques include biolistic inoculation of DNA-coated gold particles into the skin, targeting resident antigen presenting Langerhans cells (also referred to as “gene-gun” technique) [91, 102], the use of cationic poly(DL-lactide-co-glycolide) (PLG) microparticles with DNA adsorbed onto the surface [166, 167], or application of pulsed electrical fields (also referred to as electroporation in vivo) at the injection site either after i.m. or i.d. DNA administrations [168-171]. It is worthwhile to note here that direct injection of naked DNA into a peripheral lymph node was shown to induce strong CTL responses, which were qualitatively and quantitatively superior to that achieved by conventional i.m. or i.d. inoculation routes [172]. This finding suggests that efficacy of priming of naive T cells after DNA immunization correlates with the strength and duration of antigenic stimulus in secondary lymphoid organs.
The immunogenicity of DNA vaccines can also be enhanced by various modifications of the plasmid-encoded antigens. Codon optimization of the encoding DNA sequences has been shown to increase antigen expression resulting in superior antibody and CTL responses after DNA vaccination [173, 174]. Linking of the antigen to a ubiquitin monomer [105] or heat shock proteins [106, 175] enhanced antigen-specific CTL responses, presumably via improved targeting of these fusion proteins to the conventional or cross-priming MHC class I presentation pathways.
Another strategy to optimize induction of immune responses by DNA vaccines is based on the fact that induction of helper CD4+ T cells significantly contributes to the generation of effector CTL and antibody responses (see section 4.1). Providing CD4+ T cell help by means of linkage of a tumor antigen with a microbial or viral antigen, containing strong helper epitopes, was shown to result in enhanced antibody and CTL responses against the tumor antigen after DNA vaccination (reviewed in [176]).
It is important to mention that design of such tumor antigen—“helper” antigen fusion constructs, with the aim to enhance tumor antigen-specific CTL responses, requires an additional consideration. A naturally occurring focusing of CTL responses onto a very few peptide epitopes from a large antigen, known as the phenomenon of immunodominance, is observed also with DNA vaccines [177, 178]. Thus, in order to ensure that the CTL response develops against tumor antigen-derived epitopes rather than “helper” antigen-derived ones, all potential CTL epitopes in the “helper” portion of the fusion should be removed. [179, 180].
Although, recent experiments in TLR9−/− and MyD88−/− mice have demonstrated that activation of APCs by the plasmid DNA backbone is not absolutely required for induction of immune responses [123, 124], the CpG-mediated stimulation of APCs could provide certain adjuvant effects for DNA vaccines. The CpG motifs provided in the form of synthetic oligodeoxynucleotides (CpG-ODNs) were shown to act as adjuvants promoting better antibody and CTL responses after DNA vaccination (reviewed in [181]). The adjuvant effect of CpG-ODNs is very profound in combination with low DNA vaccine doses, but only modest with higher doses of DNA vaccine [182]. It is important to emphasize, that the CpG-ODNs providing optimal immunostimulatory activity in mice differ in sequence from those functioning in primates [183].
Several other strategies for enhancing the potency of DNA vaccines have focused on the use of various immunostimulatory molecules including cytokines and costimulatory molecules (reviewed in [184, 185]). These adjuvants can be administered in the form of recombinant proteins or as a separate plasmid encoding the selected molecule. The rationale behind such approaches is commonly based on facilitating priming of T cells by providing additional signals through cytokine/costimulatory molecules, which otherwise might not be optimal when using plasmid DNA vaccines alone. Examples of successful application of this approach include enhanced antibody and CTL responses leading to better protection against tumor challenge in mice immunized with CEA-encoding plasmid together with IL-12 expressing plasmid [186], and enhanced antibody/CTL responses after co-administration of an antigen-encoding DNA vaccine and plasmid expressing murine granulocyte-macrophage colony-stimulating factor (GM-CSF) [187], [188].
While all of the above-mentioned strategies were generally shown to increase immunogenicity of DNA vaccines encoding model antigens, no selected strategy is yet firmly established to provide better priming of CTL or antibody responses after DNA immunization against poorly immunogenic “self” tumor antigens in appropriate murine models and more importantly in clinical settings.
DNA Vaccines Against Cancer in Clinical Trials
Here we discuss several recently conducted Phase I clinical trials on DNA vaccination targeting tumor-associated antigens in patients with HPV-associated anal dysplasia [213], metastatic colorectal carcinoma [212], B-cell lymphoma [214], metastatic melanoma [215, 216] and prostate cancer [211, 217]. A number of different DNA delivery techniques and adjuvants were employed in these studies, which well represent current advances within the DNA vaccination field.
A standard dose escalation scheme was followed in most of these trials, with no DNA vaccine dose escalation in individual patients. DNA vaccination was applied as monotherapy and the patients had not undergone any other form of therapy within at least 3 weeks prior to entering trials, except for the studies in prostate cancer, where patients were concurrently receiving a hormonal therapy [217].
Due to the limited numbers of patients enrolled in these trials, the main objectives in all of the studies were to evaluate the safety of plasmid DNA administration, to monitor immune responses induced by the vaccines in a dose-dependent manner, and to assess correlation between vaccine-induced immune responses and the clinical benefits.
Collectively, these trials have shown that repetitive DNA administrations were well tolerated with no dose-limiting toxicities observed even with DNA doses reaching up to 2 mg per injection [212], demonstrating that repetitive immunizations with DNA is a safe procedure.
With regard to induction of immune responses, the “foreign” antigens were shown to be more immunogenic than the “self” tumor-associated antigens. Immunization with DNA vaccine encoding human papillomavirus E7-derived CTL epitope(s) induced T cell responses detected by IFNγ ELISPOT assay in 10 of 12 subjects [213]. A dual expression plasmid encoding CEA and hepatitis B surface antigen (HbsAg—included in the study as a control “foreign” antigen) induced HbsAg-specific antibody responses in 6 of 8 patients that were immunized repeatedly [212]. Lymphoproliferative or antibody responses against murine immunoglobulin (Ig) constant regions were also observed in 8 of 12 patients vaccinated with plasmid DNA encoding chimeric Ig molecules [214]. In contrast the rates of immune responses against autologous tumor-associated antigens were relatively low. In above-mentioned studies, CEA-specific antibody responses were not observed and only 4 of 17 patients developed lymphoproliferative responses to CEA, which showed no clear relationship to the dose or schedule of plasmid DNA immunization [212]. Similarly, only 1 of 12 patients immunized with chimeric Ig molecules developed a transient T cell response against autologous tumor-derived idiotypic (Id) determinant [214]. No CTL responses were detected against gp100-derived HLA-A2 restricted CTL epitopes in melanoma patients that were immunized with DNA encoding modified gp100 antigen [215], despite the fact that a recombinant fowl poxvirus encoding the same DNA construct was shown to induce CTL activity in 4 of 14 patients in previously performed study [218]. Transient CTL responses against a novel tyrosinase-derived HLA-A2 restricted epitope were observed overall in 11 of 24 melanoma patients, which received plasmid DNA encoding this epitope by infusions into a lymph node [216].
In the study combining repetitive administrations of a DNA vaccine and a recombinant adenovirus expressing PSMA, all patients eventually developed positive DTH response to a PSMA plasmid DNA injection, suggesting an induction of cellular immune response against PSMA, but these results were not further confirmed by other conventional in vitro assays [217].
In our recent clinical trial of DNA vaccination in patients with hormone-refractory prostate cancer, PSA-specific T cell immune responses were observed in 2 of 9 patients, with both responders being in the cohort receiving the highest DNA dose tested. [211]. A trend towards dose-dependent induction of T cell immune responses against tumor-associated antigens were observed in several of these studies [211, 214], although the epitope specificities of the reactive T cells have yet to be determined in order to firmly demonstrate presence of CTLs.
Clinical benefits of DNA vaccination as monotherapy were only modest and included: one patient with B-cell lymphoma experienced the tumor regression in bone marrow [214], three subjects with high-grade anogenital dysplasia achieved a partial histological response [213], two patients with prostate cancer exhibited stabilization of disease as judged by a decrease in serum PSA levels [211] and superior survival of the eleven melanoma patients who had detectable immune responses against tyrosinase compared with the thirteen patients who had no immune response [216]. The correlation of clinical benefits with vaccine induced-immune responses was observed only in the two latter studies [211, 216].
In summary, repetitive DNA vaccinations have shown a good safety profile in clinical settings even at high DNA doses (at 1 mg range), which seem to be required for induction of T cell immune responses in humans. The low frequency of responses may have resulted in part from the compromised immune status of the advanced stage patients enrolled in these trials. Future clinical trials can focus on patients during a remission phase or with minimal residual disease, where more pronounced clinical benefits of DNA vaccines are more likely to occur.
Xenogeneic Vaccines
It is naturally easier to induce an immune response to a foreign antigen than it is to a self antigen. For that reason, it is helpful to make an antigen as foreign as possible and still induce an immunity to the self protein. One strategy for doing this is to attach a foreign protein to a native self protein. A commonly used protein for this is keyhole lymphet cyanogen. Induction of an immune response to KLH often induces an immune response to the attached protein.
Another strategy is to use proteins similar to the native self protein but taken from another species of animals. This is called a xenogeneic protein. This has been done with a prostate antigen called prostate specific membrane antigen (PSMA). Wolchok et al used human PSMA in mice and induced a good immune response in mice to the mouse PSMA. There is currently a clinical trial using rodent xenogeneic PSMA in humans.
The strategy of using xenogeneic protein has been used in other species. Human tyrosinase was used to immunize dogs with melanoma. In general, the species have been chosen with wide differences between the species.
Publications describe the use of xenogeneic or xenogeneic antigens to induce an immune response. This includes the accidental but unrecognized use of xenoantigens such as when human PSA is used in mice. It also includes the deliberate use of xenoantigens. An example of the deliberate use of xenoantigens is the immunization of dogs with human antigens for vaccines against melanoma (Bergman et al, 2003, Long-term survival of dogs with advanced malignant melanoma after DNA vaccination with xenogeneic human tyrosinase: a phase 1 trial, Clinical Cancer Research, 9:1284-1290). The publications do not describe the use of xenogeneic proteins with minimal interspecies differences between the xenogeneic protein and the native protein to induce an optimal immune response to the native self antigen.
The human protein PSA was discovered and characterized in the 1970's. The PSA protein was first purified in 1979. Anti-rabbit serum was prepared from the protein and tissues analyzed. The human PSA was only found in prostatic tissues and not other tissues. Wang et al Purification of a human prostate specific antigen 1979, Invest. Urol. 17:159-63. Others have found human PSA in smaller amounts in other tissues. It is expressed at low levels in other epithelial like cells in lung and breast tumors. Zarghami et al Frequency of expression of prostate-specific antigen mRNA in lung tumors, 1997 μm J Clin Pathol 108(2): 184-90. Smith et al Prostate-specific antigen messenger RNA is expressed in non-prostate cells: implications for detection of micrometastases 1995 Cancer Res. 55(12): 2640-44.
The gene for human prostate-specific antigen was sequenced in 1989. Digby et al Human prostate specific antigen (PSA) gene: structure and linkage to kallifrein-like gene 1989 Nucleic Acids Research, 17(5): 2137. Klobeck et al, Genome sequence of human prostate specific antigen, 1989 nucleic Acids Research 17(10):3981
The cDNA sequence of Homo Sapiens PSA is recorded in Genebank. One sequence is listed in Genebank Accession Number AJ459783. Another was published in 1988 and submitted as Genebank Accession Number X07730 (Schultz et al. Sequence of a cDNA clone encompassing the complete mature human Prostate Specific Antigen (PSA) and an unspliced leader sequence. 1988 Nucleic Acids Research 16(13):6226. Still another human cDNA human sequence is listed as Genebank Accession Number BC056665.
The cDNA sequence of the non-human primate Cynomolgus monkey (Macaca fasicularis) prostate specific antigen precursor cDNA is published as Genebank Accession Number AY647976.
In addition, the cDNA sequence of the Rhesus monkey (Macaca mulatta) prostate specific antigen is published as Genebank Accession Number X73560.
A comparison of the preceding three genes shows that there are 30 base pair differences between human PSA and either rhesus or Cynolomogus PSA cDNA. This is a 3.8% difference. The resulting amino acid difference is 9.9% for the rhesus monkey PSA compared to human PSA and 10.7% for the Cynamologus monkey PSA compared to human.
The attached three page DNA Sequence Comparison (in FIGS. 1A, 1B, and 1C) shows the homology among the cDNA gene sequence for human PSA (cDNA on Genebank Accession # BC056665), the cDNA gene sequence for Maccaca mulatto PSA, and the cDNA gene sequence for Macca fascicula PSA.
Aside from the scientific literature discussed above, a review of published and issued patents reveal the following relevant patents and published patent application.
United States Published Application 20040141958 discloses novel methods for therapeutic vaccination and discusses the use of self peptides or proteins with foreign peptides representing CTL epitopes. This would be a chimeric protein. It does not address the use of entire foreign proteins to induce a cross reactive immunity to native proteins.
U.S. Pat. No. 5,925,362 discloses a method to elicit an antitumor response with human prostate specific antigen. This patent describes a prostate cancer vaccine requiring two parts. One is human PSA and the other is an expression system for producing the human PSA in situ. This is a DNA vaccine expressing human PSA. It does not describe the use of a xenogeneic PSA DNA vaccine.
U.S. Pat. No. 6,165,460 discloses the generation of immune responses to human prostate specific antigen (PSA). This patent describes a PSA DNA vaccine. One claim describes a pox virus vector used for expressing PSA. It does not specifically state human PSA but xenogeneic PSA is not mentioned. Another claim teaches the use of PSA (or PSA CTL epitope) followed by a second administration of additional PSA. This is a traditional vaccine boost strategy that includes boosting with a different PSA expressing vector or with PSA protein itself. A third claim mentions the use of a CTL eptiope only. Although the animal model used to evaluate the vaccine was a rhesus monkey model using human PSA, there was no mention of the opposite approach of using of rhesus monkey PSA in humans.
U.S. Pat. Nos. 6,818,751; 6,800,746; 6,759,515; 6,664,377; 6,657,056; 6,630,305; 6,620,922; 6,613,872; 6,329,505; 6,262,245; 6,261,562; 5,854,206 all describe the use of prostate specific peptides for diagnosis of prostate cancer, generation of monoclonal antibodies against the peptides, and immunotherapy of prostate cancer. They do not discuss the use of xenogeneic PSA as an immunogen.
U.S. Pat. No. 6,699,483 discloses cancer treatments which employ the use of three human prostate cancer cell lines in a prostate cancer vaccine. The cell lines are human and represent a broad range of antigens. It does not describe the use of xenogeneic cell lines, xenogeneic PSA or the use of DNA vaccines.
From the above discussion, a wide variety of approaches have been discussed in the scientific literature relating to vaccines, including xenogeneic approaches.
For purposes of the present invention, a brief review of relevant points is provided.
It is known that when a non-human antigen is introduced into a human, a human immune response produces antibodies against the non-human antigen.
It is believed that, in certain limited specific cases, when a specific-case non-human antigen is introduced into a human, a human immune response produces antibodies against a similar specific-case human antigen.
Only primates have prostate specific antigen (PSA). Humans have human-PSA, and non-human primates have non-human-primate-PSA. Species such as mice and dogs have serine proteases that are kallikrien proteins, but they are different enough from PSA so they are not considered to be PSA.
Non-human primates include the rhesus monkey and the chimpanzee, among others.
Human-PSA is comprised of a sequence of approximately 260 amino acids (See FIG. 2, section “huPSA”). Rhesusy-monkey-PSA is comprised of a sequence of approximately 260 amino acids (See FIG. 2, section “rhPSA”). Approximately 10% of the amino acids in the rhesus-monkey-PSA amino acid sequence differ from the amino acids in the human-PSA amino acid sequence.
Though there are similarities between human-PSA and xenogeneic-PSA, such as Rhesus-monkey-PSA, the differences between the human-PSA and the Rhesus-monkey-PSA are so significant that laboratory tests for detection of human-PSA will not detect xenogeneic-PSA such as Rhesus-monkey-PSA.
Turning specifically to prostate cancer in humans, treatment of human prostate cancer involves surgically removing the entire prostate gland. Further treatment of prostate cancer involves trying to destroy prostate cells that escaped surgical removal. In this respect, an anti-prostate vaccine should be designed to kill all prostate cells that escaped surgical removal.
Human prostate cells produce human-PSA, and a vaccine against human prostate cancer should cause triggering a human immune response that brings about the killing of human cells that produce human-PSA. In this way, prostate cells that escaped surgery would be killed as a result of vaccination with the vaccine against human prostate cancer.
In view of the prior art, it is an insight of the present inventors, resulting in the present invention, that it would be desirable to use a xenogeneic antigen (e.g. protein) in a human, wherein, with respect to the xenogeneic antigen that is used, there are relatively few interspecies differences between the xenogeneic antigen and the human self antigen in order to induce an optimal immune response in the human to its native self antigen.
Another insight of the present inventors, resulting in the present invention, is that it would be desirable to use a non-human primate xenogeneic antigen (e.g. protein) in a human, wherein, with respect to the non-human primate xenogeneic antigen that is used, there are relatively few interspecies differences between the non-human primate xenogeneic antigen and the human self antigen in order to induce an optimal immune response in the human to its native self antigen.
A more specific insight of the present inventors, resulting in the present invention, is that it would be desirable to use a non-human primate xenogeneic PSA antigen in a human, wherein, with respect to the non-human primate xenogeneic PSA antigen that is used, there are relatively few interspecies differences between the non-human primate xenogeneic PSA antigen and the human self PSA antigen in order to induce an optimal immune response in the human to its native self PSA antigen.
An even more specific insight of the present inventors, resulting in the present invention, is that it would be desirable to provide a method for inducing an immune response against human PSA in humans using a non-human PSA having an amino acid homology ≧88% and ≦98% (or a 2 to 12% difference) with respect to the human PSA.
More specifically, the subject method includes obtaining PSA isolated from non-human primates and molecularly altering the non-human-primate PSA to adjust the amino acid homology to an optimal homology balance with respect to the human PSA. More specifically, it is desirable to provide an optimal homology balance such that the amino acid sequence in the non-human-primate PSA has a homology that is different enough from the amino acid sequence of the human PSA to induce an immune response to the non-human-primate PSA in the human but similar enough to produce an immune response in the human to human PSA.
The present invention also includes a DNA sequence whereby expression of that sequence produces a protein with an amino acid sequence with 88 to 98% homology to human PSA. It also includes the use of the DNA sequence in a polynucleotide vaccine such as a DNA or RNA vaccine.
A rationale for using non-human primate PSA rather than artificially made PSA is that all of the changes in the natural selection of a protein that maintains serine protease activity selects proteins with similar conformation. This means that immune responses to areas of the protein that are conformationally dependent are more likely to be similar or the same among the human and primate xenogeneic PSA.
In accordance with the present invention, delivery of the vaccine can be by a variety of methods including injection with or without chemical enhancers of transfection, biolistic methods, or electroporation for example.
Thus, while the foregoing body of prior art indicates it to be well known to use xenogeneic antigens for eliciting some immune responses in non-human animal models, the prior art described above does not teach or suggest methods and compositions relating to a vaccine against prostate cancer which has the following combination of desirable features: (1) an anti-prostate vaccine which is designed to kill prostate cells that have escaped surgical removal; (2) causes a triggering of a human immune response that brings about the killing of human cells that produce human-PSA; (3) is not limited to hormonal therapies; (4) uses a xenogeneic antigen (e.g. protein) in a human, wherein, with respect to the xenogeneic antigen that is used, there are relatively few interspecies differences between the xenogeneic antigen and the human self antigen in order to induce an optimal immune response in the human to its native self antigen; (5) uses a non-human primate xenogeneic antigen (e.g. protein) in a human, wherein, with respect to the non-human primate xenogeneic antigen that is used, there are relatively few interspecies differences between the non-human primate xenogeneic antigen and the human self antigen in order to induce an optimal immune response in the human to its native self antigen; and (6) uses a non-human-primate xenogeneic PSA antigen in a human, wherein, with respect to the non-human-primate xenogeneic PSA antigen that is used, there are relatively few interspecies differences between the non-human-primate xenogeneic PSA antigen and the human self PSA antigen in order to induce an optimal immune response in the human to its native self PSA antigen. The foregoing desired characteristics are provided by the unique methods and compositions relating to a vaccine against prostate cancer of the present invention as will be made apparent from the following description thereof. Other advantages of the present invention over the prior art also will be rendered evident.